Transplant recipients developing anti-donor HLA antibodies after transplantation demonstrate increased risk of antibody-mediated rejection, transplant associated vasculopathies and graft loss. HLA class I signaling pathways have been implicated in these processes because ligation of class I molecules by anti-HLA antibodies initiates intracellular signals in endothelial cells that synergize with growth factor receptors to elicit survival and proliferation. We found that anti-HLA antibodies elicit different signal transduction outcomes, survival vs. proliferation, depending upon their specificity, titer and the degree of HLA antigen expression on the endothelial cell. These studies provide a paradigm for further elucidating the molecular mechanisms underlying how the concentration of antibody mediates survival and proliferation events in endothelium and their relationship to transplant outcome. Activation of anti-apoptotic and cell survival machinery in endothelial cells is augmented when cells are exposed to low concentrations of anti-HLA antibodies. In contrast, treatment of endothelial cells with high concentrations of HLA antibodies stimulates cell proliferation. This suggests that low levels of antibody binding to HLA may be beneficial to transplant survival by activating survival pathways that promote graft accommodation. On the other hand, high levels of antibody binding may have a detrimental effect on graft survival by upregulating FGFR expression, stimulating cell proliferation and increasing risk for development of transplant vasculopathy. We believe these findings are clinically relevant and may explain differences in transplant outcome in recipients producing anti-donor HLA antibodies. The overall goals of this proposal are to elucidate whether the intracellular signaling events initiated by antibody ligation of class I molecules are influenced by the specificity and concentration of the antibody and to determine the clinical relevance of class I signaling pathways in transplantation. Aim 1 will focus on HLA interactions with 2 integrins and survival and proliferation phosphorylation cascades induced by different titers of anti-HLA antibody on primary human endothelial cells. These studies will permit us to dissect the class I signaling pathways and explore the cause-effect relationships between proteins in the pathway. Aim 2 will test whether the antibody induced phosphorylation cascades identified in cultured EC are also operational in vivo. We will study the effect of different titers and specificities of anti-MHC antibody on induction of cell survival and proliferation signal transduction pathways in a RAG1 knock out murine heterotopic cardiac transplant model. Aim 3 will explore whether anti-MHC class I induced phosphorylation of proteins in cardiac transplant biopsies correlate with the presence and titer of circulating anti-donor-HLA antibodies, diagnosis of antibody-mediated acute and chronic rejection and transplant outcome. These experiments will confirm the biological relevance of class I mediated signaling in human transplantation.